The construction of an infectious clone of grapevine virus A (GV A)

Hele tekst

(1)THE CONSTRUCTION OF AN INFECTIOUS CLONE OF GRAPEVINE VIRUS A (GVA). by Jacques du Preez. Presented in partial fulfillment of the requirements for the degree of Master of Science at the Department of Genetics, University of Stellenbosch.. April 2005 Supervisor: Prof JT Burger.

(2) Declaration I, the undersigned, hereby declare that the work contained in this thesis is my own original work and that I have not previously in its entirety or in part submitted it at any university for a degree.. ____________________. Date: ________________. J. du Preez. ii.

(3) Abstract An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA-mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch' s postulates will also have to be proved in order to confirm iii.

(4) the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.. iv.

(5) Opsomming ‘n Infektiewe kloon is ‘n cDNA kloon van ‘n virale RNA genoom wat in vitro of in vivo getranskribeer kan word. Infektiewe klone dien as hulpmiddel om virale RNA genome op molekulêre vlak te bestudeer om sodoende dieper insig te verkry oor genoomorganisasie, virale geenfunksie, teenwoordigheid van regulatoriese volgordes en geenuitdrukking. In die Wes-Kaap (onder andere) is ‘n nuwe kruppelende wingerdsiekte, bekend as Shiraz siekte, besig om pos te vat. Die etiologie van die siekte en patogeniese agense daarby betrokke word nog nie ten volle verstaan nie. Studies toon dat Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, met die siekte-toestand geassosieerd is. Die doel van hierdie studie was om ‘n vollengte infektiewe cDNA kloon van GVA te konstrueer om sodoende te dien as instrument in die molekulêre studie van die virusgenoom. Hierdie kloon kan in die toekoms ingespan word om GVA se betrokkenheid by Shiraz siekte te bestudeer, wat weer kan lei tot die ontrafeling van die etiologie en beheer van die siekte. ‘n Vollengte kloon van GVA, genaamd GVA-IC2/T7-2972-3,. was. in. verskeie. stappe. gekonstrueer. deur. gebruik. te. maak. van. beperkingsvertering/ligasie en inleier oorvleuelings-verlenging PCR. Grapevine virus A cDNA fragmente is vanuit GVA-geïnfekteerde N. benthamiana en Vitis vinifera plante verkry deur van drie verskillende tegnieke gebruik te maak, waarvan die vinnige direkte enkel-buis RT-PCR die suksesvolste was. ‘n 5’ T7 promotor en ‘n 3’ poli-A stert is geïnkorporeer en die vollengte kloon is in pBluescript II SK (+) gekloneer. Vollengte volgordebepaling van die kloon het twee beduidende leesraam-verskuiwingsmutasies getoon. Die eerste mutasie was ‘n enkel basispaar invoeging (een G) in ‘n “glyende setel” van ses G’s by posisie 1380-1385 in oop leesraam een (ORF1) van die virusgenoom. Hierdie mutasie is met PCR-gebaseerde setelgerigte mutagenese herstel, wat gelei het tot pSK-GVAmutagen-3 en pSK-GVA-mutagen-4. Die tweede mutasie was ‘n enkel basispaar delesie (een G) by posisie 6959 in ORF4, wat vir die kapsiedproteïen (CP) kodeer. Verskeie tegnieke is aangepak om dié mutasie te herstel, maar geen was suksesvol nie. Alhoewel die tweede mutasie nie herstel kon word nie, is in vitro transkripsies gedoen op die drie klone, gevolg deur die onderskeie infeksies van N. benthamiana plante. Die drie klone het pSK-GVA-mutagen-3, pSK-GVA-mutagen-4 (wat die 6959 mutasie bevat) en GVA-IC2/T7-2972-3 (wat beide mutasies bevat), ingesluit. Geen beduidende visuele simptome is teen dag 21, post-innokulasie, waargeneem in plante wat geïnfekteer is met in vitro RNA of in plante geïnfekteerd met wilde tipe virus nie. Vinnige direkte enkel-buis RT-PCR resultate het die teenwoordigheid van virale RNA in geïnfekteerde en apikale blare van geïnfekteerde plante getoon, en voorlopige bewyse gelewer dat die gemuteerde klone op een of ander manier in staat is tot sistemiese v.

(6) infeksie en virale beweging. Hierdie resultate is steeds voorlopig en verskeie post-infeksie studies moet nog gedoen word om hierdie bevindings te bevestig. Koch se postulate sal ook moet bewys word om die infektiewe status van die klone te bevestig. Die effek van die twee mutasies in die gekonstrueerde klone sal verder getoets moet word en post-infeksie analises gedoen word om te ondersoek of die virusnageslag mutasie-vry is. Drie vollengte GVA cDNA klone (bevattende mutasies) wat skynbaar die vermoë het om N. benthamiana plante sistemies te infekteer, is in hierdie studie gekonstrueer. Dit het die grondslag gelê vir molekulêre en mutasie-analise van die GVA genoom. Dit kan uiteindelik lei tot die studie van patogeen-gasheer interaksies om die etiologie van Shiraz siekte te ontrafel.. vi.

(7) Abbreviations A600 A Amp ATP bp β-ME BMV BNYVV BSA C CaMV cDNA CP CTAB CTV DI RNA DNA dNTPs dRNA DTT ddH2O dsRNA EDTA ELISA EtOH g G GDP GES GFP GGP GLRaV-3 GVA GVB GVC GVD h HLV IEM IPTG kb kDA LB MOPS MP. Absorpsion value at 600nm Adenine Ampicillin Adenosine Triphosphate Base pairs β-Mercaptoethanol Brome mosaic virus Beet necrotic yellow vein virus Bovine Serum Albumin Cytosine Cauliflower mosaic virus Complementary deoxyribonucleic acid Coat protein N-Cetyl-N,N,N-trimethyl Ammonium Bromide Citrus tristeza virus Defective interfering ribonucleic acid Deoxyribonucleic acid Deoxynucleoside triphosphate(s) Defective ribonucleic acid Dithiothreitol Double distilled water Double stranded ribonucleic acid Ethylene Diamine Tetra-acetic Acid di-sodium salt Enzyme-Linked Immunosorbent Assay Ethanol Gram(s) Guanine Gross domestic product Glysine-NaOH/EDTA/Sodium Green Fluorescent Protein Gross geographic product Grapevine leafroll-associated virus 3 Grapevine virus A Grapevine virus B Grapevine virus C Grapevine virus D Hour(s) Heracleum latent virus Immuno-electronmicroscopy Isopropylthio- -D-galactoside Kilobase Kilo Dalton Luria Berthani broth (3-[N-morpholino]propanesulfonic acid) Movement protein vii.

(8) µg µl µM M min ml mM mRNA NaOAc nt ORF PCR PEG PIPES PTGS RE RSP PVA PVP PVY RdRp RNA rpm RT RT-PCR Sec sgRNA ssRNA SD SDS SSCP STE T TAE Tris U U UV UTR V v\v w\v X-Gal. Microgram(s) Microliter Micromolar Molar Minute Millilitre(s) Millimolar Messenger ribonucleic acid Sodium acetate Nucleotide Open reading frame Polymerase Chain Reaction polyethylene glycol 1,4-Piperazinediethanesulfonic acid Post-transcriptional gene silencing Restriction endonuclease Rupestris stem pitting virus Potato virus A Polyvinyl pyrrolidone Potato virus Y RNA-dependant RNA polymerase Ribonucleic Acid Revolutions per minute Reverse transcription Reverse Transcription-Polymerase Chain Reaction Second(s) Sub-genomic ribonucleic acid Single stranded ribonucleic acid Shiraz disease Sodium Dodecyl Sulphate Single-strand conformational polymorphisms Sodium/Tris/EDTA Thymine Tris/acetic acid/EDTA Tris(hydroxymethyl)aminomethane Unit(s) Uracil Ultra Violet Un-translated region Volt Volume per volume Weight per volume 5-bromo-4-chloro-3-indocyl- -D-galactoside. viii.

(9) Acknowledgements I would like to express my sincerest gratitude to the following : •. My supervisor, Prof JT Burger for his support, guidance and encouragement throughout this study.. •. Michael-John Freeborough for his advice, encouragement, support and interest in my work.. •. Julia Robson, Hano Maree and Chris van Eeden for their technical assistance and advice.. •. Harry Crossley Foundation, National Research Foundation, THRIP, Winetech and the University of Stellenbosch for their financial support.. •. The Vitis lab: Anthony, Beverly, Chris, Hano, Helena, Julia, Mandi, Michael-John, Mike and Ndiko for creating an enjoyable working environment.. •. My friends and family.. ix.

(10) In questions of science the authority of a thousand is not worth the humble reasoning of a single individual. -Galileo Galilei. Of course, if one ignores contradictory observations, one can claim to have an 'elegant' or 'robust' theory. But it isn't science. - Halton Arp, 1991, from Science News, Jul 27.. x.

(11) Contents Abstract Opsomming Abbreviations Contents 1. Introduction. 1. 1.1. GENERAL INTRODUCTION. 1. 1.2. PROJECT PROPOSAL. 3. 2. Literature review. 4. 2.1. INTRODUCTION. 4. 2.2 GRAPEVINE VIRUS A (GVA). 6. 2.2.1. Genus Vitivirus. 6. 2.2.2. Morphology. 6. 2.2.3. Genome and Genomic organization. 7. 2.2.4. Transmission. 8. 2.2.5. Diseases and Geographical distribution. 9. 2.2.5.1. Shiraz disease and Syrah decline. 10. 2.2.5.1.1. Shiraz disease (SD). 10. 2.2.5.1.2. Syrah decline. 11. 2.2.6. Molecular diversity. 11. 2.2.7. Replication mechanism of GVA. 13. 2.2.7.1. Synthesis of Subgenomic RNAs by Positive-Strand RNA viruses. 15. 2.2.7.2. Subgenomic RNAs in GVA?. 15. 2.2.8. Defective RNAs. 16. 2.2.9. RNA gene silencing. 16. 2.3. INFECTIOUS CLONES 2.3.1. Construction of infectious clones 2.3.1.1. Full-length cDNA clones 2.3.1.1.1. Basic approach in construction of full-length cDNA clones 2.3.1.2. Infectious cDNAs 2.3.1.2.1. Advantages 2.3.2. cDNA clones for plant RNA viruses 2.3.2.1. Infectious RNA transcripts from Grapevine virus A cDNA clone 2.3.3. Factors influencing infectivity of the cDNA clone. 17 18 18 18 19 19 19 20 20. 2.3.3.1. The heterogeneity of transcript population. 21. 2.3.3.2. The presence of point mutations. 21. xi.

(12) 2.3.3.3. Presence of nonviral nucleotides. 21. 2.3.3.3.1. Effect of 3’-extensions. 22. 2.3.3.3.2. Effect of 5’-extensions. 22. 2.3.3.3.3. Effect of the cap structure. 23. 2.3.3.4. Instability in bacteria. 23. 2.3.3.5. RNA polymerases. 24. 2.3.4. Conclusion. 24. 3. Materials and Methods. 25. 3.1. PLANT MATERIAL. 25. 3.1.1. Plant material. 25. 3.1.2. Plant cultivation. 25. 3.2. GENERATION OF GVA cDNA FRAGMENTS. 25. 3.2.1.. Double stranded RNA (dsRNA) extraction: CFII cellulose method. 25. 3.2.2. Total RNA extraction. 26. 3.2.3. Primer design. 26. 3.2.4. Reverse transcription polymerase chain reaction (RT-PCR). 28. 3.2.4.1. Primer annealing. 28. 3.2.4.2. cDNA synthesis. 28. 3.2.4.3. PCR amplification. 29. 3.2.5. Rapid direct-one-tube RT-PCR. 30. 3.3. AGAROSE GEL ELECTROPHORESIS. 30. 3.4. PCR PRODUCT GEL PURIFICATION. 30. 3.5. DNA QUANTIFICATION. 31. 3.6. CLONING OF PURIFIED PCR PRODUCTS INTO PGEM -T EASY VECTOR. 31. 3.6.1. Ligation systems. 31. 3.6.2. Preparation of ultracompetent cells: Rubidium chloride. 32. 3.6.3. Escherichia coli transformation. 33. 3.6.4. Screening for positive white colonies with colony PCR. 33. 3.6.5. Plasmid DNA purification. 33. 3.7. FREEZER CULTURES. 34. 3.8. SEQUENCING OF CLONES. 35. 3.9. SEQUENCE ANALYSIS. 35. 3.10. JOINING OF OVERLAPPING CLONES. 35. 3.10.1. Restriction digestion and ligation of inserts and vectors. 35. 3.10.2. Primer Overlap Extension PCR. 36. 3.10.2.1. First amplification. 37. 3.10.2.2. Second amplification. 38. xii.

(13) 3.11. INCORPORATION OF THE 3’ POLY-A TAIL. 38. 3.12. INCORPORATION OF THE 5’ T7 PROMOTER. 39. 3.12.1. Incorporation of the T7 using the Geiser Technique. 40. 3.12.1.1. Expand Two-step Geiser High Fidelity PCR. 40. 3.12.1.2. Dpn I digestion. 40. 3.12.1.3. Purification of digestions. 40. 3.12.1.4. Transformation. 40. 3.12.2. Incorporation of the T7 promoter using Expand High Fidelity PCR. 41. 3.13. PCR-BASED SITE-DIRECTED MUTAGENESIS. 42. 3.14. IN VITRO TRANSCRIPTION. 42. 3.15. INFECTION OF N. BENTHAMIANA. 42. 4. Results and Discussion. 44. 4.1.. AMPLIFICATION OF GVA cDNA FRAGMENTS. 44. 4.1.1. Plant material. 44. 4.1.2. Total RNA extraction followed by RT-PCR. 45. 4.1.3. dsRNA extraction followed by RT-PCR. 46. 4.1.4. Rapid Direct-One-Tube-RT-PCR. 46. FULL-LENGTH GVA CONSTRUCT ASSEMBLY FROM TWELVE SELECTED FRAGMENTS. 49. 4.2.1. Step 1: Joining of fragments GVA-5’-620 and GVA-924 by restriction digestion. 52. 4.2.2. Step 2: Joining of fragments GVA-1005 and GVA-951 by restriction digestion. 53. 4.2.3. Step 3: Joining of fragments GVA-908 and GVA-1099 by restriction digestion. 54. 4.2.4. Step 4: Joining of fragments GVA-532 and GVA-759 by primer overlap extension. 55. 4.2.5. Step 5: Joining of fragments GVA-965s and GVA-1002s by primer overlap extension. 56. 4.2.6. Step 6: Joining of fragments GVA-1746s and GVA-501s by primer overlap extension. 58. 4.2.7. Step 7: Joining of fragments GVA-1694s and GVA-646 s by primer overlap extension. 59. 4.2.8. Step 8: Joining of fragments GVA-1188 and GVA-1271s by primer overlap extension. 60. 4.2.9. Step 9: Joining of fragments GVA-646s and GVA-1884 by primer overlap extension. 61. 4.2.10. Step 10: Generation of fragments GVA-1558 and GVA-1418 by primer overlap extension. 62. 4.2.11. Step 11: Joining of fragments GVA-2193 and GVA-1558 by restriction digestion. 66. 4.2.12. Step 12: Joining of fragments GVA-1418 and GVA-1673 by restriction digestion. 69. 4.2.13. Step 13: Joining of fragments GVA-2193/1558 and GVA-1601 by restriction digestion. 69. 4.2.14. Step 14: Incorporation of the 3' -poly-A tail. 70. 4.2.15. Step 15: Incorporation of the 5' -T7 promoter. 72. 4.2.16. Step 16: Cloning of fragment GVA-T7-3794 into pBluescript II SK (+). 72. 4.2.. 4.2.17. Step 17: Cloning of Aat II/Sal I digested pGEM-GVA-2308-polyA into Aat II/Sal I digested pSK-GVA-T7-3794. 72. 4.2.18. Step 18: Cloning of Aat II digested pGEM-GVA-1418/1673 into Aat II linearized pSKGVA-T7-5' -3'. 73. xiii.

(14) 4.3. CORRECTION OF MUTATIONS IN FIVE FULL-LENGTH CLONES 4.3.1. Incorporation of the T7 promoter with Expand High Fidelity PCR. 74 74. 4.3.2. Cloning of Ksp I and Nar I digested T7-2972-3 into Ksp I and Nar I digested pSK-GVAIC2, pSK-GVA-IC3, pSK-GVA-IC5, pSK-GVA-IC6, and pSK-GVA-IC8. 4.3.3. Correction of the mutation at position 1380 – 1385 4.3.3.1. PCR-based site-directed mutagenesis. 74 76 76. 4.3.4. Cloning of Ksp I and Nar I digested pGEM-mutagen-1 and pGEM-mutagen-2 into Ksp I and Nar I digested pSK-GVA-IC2/T7-2972-3. 4.3.5. Correction of the mutation at position 6959. 78 78. 4.4. IN VITRO RNA TRANSCRIPTION. 80. 4.5. INFECTION OF N. BENTHAMIANA. 80. 4.5.1. Symptom development in infected plants. 81. 4.5.2. RT-PCR screening of infected plants. 81. 4.6. GENERAL DISCUSSION. 82. 5. Conclusion. 86. Appendix 1. 88. Appendix 2. 91. Appendix 3. 93. Appendix 4. 109. 6. References. 117. xiv.

(15) Chapter 1: Introduction. 1.1. GENERAL INTRODUCTION The wine industry plays an important role in the South African economy and contributes 3% to the South African gross domestic product (GDP) (World development report, 2003). According to the Wines of South Africa statistics report in 2003 (http://www.wosa.co.za), there are currently 110 200 hectares of vines producing wine grapes, under cultivation in South Africa. Red varietals account for approximately 45% of the national vineyard, with Cabernet Sauvignon representing 15% and Shiraz 9% of the total. Due to the growth in red wine consumption and shifting market demands, the industry is rapidly increasing its plantings of red wine varietals with Shiraz showing the most dramatic growth. Shiraz was the most planted variety in 1999 and 2000. In 2003 South Africa produced an annual harvest of 965m litres, of which 75% was devoted to the making of premium wine. South Africa is the ninth ranked wine producing country worldwide, producing 3.1% of the world' s wine. In the 2003 calendar year, 237.3m litres of natural bottled wines were exported by South Africa, showing an increase of 10% on the previous year. Furthermore, red wine exports grew by 13% to account for 45% of all natural wines exported. In the case of bottled wines, Shiraz, Merlot, Chardonnay and Sauvignon Blanc varietals showed the most export growth in 2003 compared to the previous year. The wine industry currently directly or indirectly employs 348 500 people, including farm labourers, those involved in packaging, retailing and wine tourism. Wine tourism employs some 48 350 of these people. A study conducted by the SA Wine Industry Information & Systems (SAWIS) showed that in 1999, the wine industry contributed 9.7% to the Western Cape' s gross geographic product (GGP). This study also concluded that some R3.5 billion of the R14.6 billion contributed by the wine industry to the regional economy was generated indirectly through wine-tourism activities centered in the winelands (http://www.sawis.co.za). These statistics indicate that it is important to protect this resource so that the economy of the Western Cape, and ultimately South Africa, is not negatively affected. As with all crop plants, grapevines are susceptible to disease causing organisms and pests, which include, bacteria, fungi, insects, nematodes, phytoplasmas and viruses. Of these pests and pathogens, viruses are some of the most devastating pathogens over the long term, causing losses of millions of Rands, although it is difficult to make a formal estimate. Virus control is made difficult by the fact that 1.

(16) there are no cures or treatments, and furthermore no grapevine resistance genes or other natural resistance genes have been discovered against grapevine viruses. It has been suggested that GVA is currently the second most important virus in South African vineyards, next to Grapevine leafroll-associated virus 3 (GLRaV-3), genus Closterovirus, family Closteroviridae. It has a negative impact on South African vineyards, for it is associated with two destructive diseases namely, grapevine leafroll disease and Shiraz disease (Pietersen, G., Pers. Comm., Department of Plant Pathology, University of Pretoria, South Africa). The main interest in GVA is its role in Shiraz disease, as Shiraz is one of the most economically important red cultivars in South Africa. Shiraz disease is an emerging disease of grapevine in the Western Cape (and elsewhere) with very distinctive symptoms, and has a crippling effect in vineyards. This deadly disease infects the noble grapevine cultivars Shiraz and Merlot in South Africa. Infected vines deteriorate relatively fast and normally die within five years. The aetiology of the disease is largely unknown, but is believed to be of multiple viral origin with GVA consistently associated with the disease (Goszczynski, D., Pers. Comm., Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). In order to elucidate disease and ultimately control viruses in South African vineyards, research needs to be performed on important grapevine diseases and their agents. Grapevine virus A is the most likely agent of Shiraz disease. Grapevine virus A has a single stranded RNA genome, which makes it difficult to study at a molecular level. Viral genomes are amenable to investigations into their organization and expression, by recombinant DNA technology, because of their small size. Deeper insight can be gained by analyzing and modifying viral genomes at a molecular level. The potential of investigations has greatly been enhanced by the possibility of obtaining infectious clones (as in vitro-transcribed RNA copies or as cDNAs) corresponding to the genomes of RNA viruses (Boyer & Haenni, 1994). This will serve as a powerful tool to study RNA viral genomes at the molecular level to gain insight into gene organization and replication of RNA viruses. This will lead to the unravelling of gene expression and pathogen-host interactions to ultimately develop resistance, which will lead to the elucidation of disease.. 2.

(17) 1.2. PROJECT PROPOSAL. The aim of this study was to create a tool to investigate GVA’s role in Shiraz disease and to study pathogen-host interactions on a molecular level. We aimed to construct a full-length infectious cDNA clone of the virus in order to ultimately develop a transient expression vector for grapevine in order to assist in elucidating the aetiology of this disease, and possibly obtaining resistance to Shiraz disease. The full-length infectious clone can shed light on the genome organization and gene expression of GVA.. Specific tasks/steps of this study were: •. Design of GVA-specific primers. •. Total RNA extraction or dsRNA extraction from GVA-infected grapevine or tobacco or direct onestep RT-PCR on GVA-infected grapevine or tobacco. •. Cloning of GVA PCR fragments into pGEM -T Easy Vector. •. Sequencing of cloned fragments. •. Construction of a full-length cDNA clone by joining of overlapping cloned fragments by restriction digestion and ligation or primer extension PCR. •. Incorporation of a T7 phage promoter upstream of the 5’-end of GVA. •. Incorporation of a poly-A tail downstream of the 3’-end of GVA. •. Cloning of the full-length clone into pBluescript II SK (+). •. In vitro RNA transcription followed by infection of tobacco. •. Post-infection analysis. 3.

(18) Chapter 2: Literature Review 2.1. INTRODUCTION Shiraz (known as Syrah elsewhere) is one of the most important red grapevine cultivars worldwide. The wine industry in South Africa is drastically increasing its plantings of Shiraz in order to keep up with the growth in red wine consumption (http://www.sawis.co.za). An increase in produce is hampered by the fact that a new disease is emerging in the Western Cape (and elsewhere), known as Shiraz disease. This disease, of which the aetiology is largely unknown, affects the noble grapevine cultivars of Shiraz and Merlot in South Africa. It causes infected grapevines to deteriorate very fast, show distinctive crippling symptoms and eventually die within five years (Goszczynski, D., Pers. Comm., Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). It is believed that multiple viruses are associated with the disease. A recent study undertaken by Goszczynski et al. (2003) revealed that GVA is consistently associated with the disease (Goszczynski, D., Pers. Comm., Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). Another distinct new emerging disease, known as Syrah decline, is affecting Syrah vineyards in France and California. As with Shiraz disease, very little is known about this mysterious condition and care should be taken not to confuse these two distinct conditions with each other. To further complicate matters, the symptoms of Syrah decline in France and California differ drastically from each other. In France the vines eventually die, whereas in California this condition does not lead to death. For this reason, the condition in California was provisionally called Syrah disorder (Stamp, 2004). Grapevine virus A, consistently associated with Shiraz disease, is a member of the recently established genus Vitivirus (Martelli et al., 1997). It is a phloem-limited virus (Minafra et al., 1997), with a linear, positive sense single-stranded (ss) RNA genome (www.dpvweb.net/dpv/showdpv.php?dpvno=383). The complete nucleotide sequence of GVA has been determined (Minafra et al., 1994; 1997) and it was found that the genome is translated by means of five open reading frames (ORF) (www.dpvweb.net/dpv/showdpv.php?dpvno=383). In 1999, the construction of an infectious clone of GVA isolate PA3 was reported by Galiakparov et al. Mutation analysis was performed on the clone to experimentally define the role of every GVA gene. Putative translation products were assigned for every ORF except for ORF 2 (Galiakparov et al., 2003). Utilization of the clone to unravel the 4.

(19) regulation of gene expression and the synthesis of viral sgRNAs in GVA, led to the characterization of two nested sets of sub-genomic RNAs (sgRNAs) (Galiakparov et al., 2003). sgRNA-producing viruses are used increasingly for expression of vaccines and other pharmaceutically useful proteins in plants (McCormick et al., 1999). Insight can be gained into the origin and diversification of viruses by recombination and RNA virus replication if the mechanism of sgRNA synthesis is fully understood. The possibility that defective RNAs may arise during vitivirus replication was investigated by Obreque et al., (2003). This led to the detection of deleted chimaeric RNAs, with conserved 5’ and 3’ ends, resembling defective RNAs in GVA-infected N. benthamiana plants. Recently, clear-cut posttranscriptional gene silencing (PTGS) suppression was observed in N. benthamiana transgenic plants, expressing a GFP (green fluorescent protein) transgene, following GVA infection. Identification of the GVA protein(s) determining PTGS suppression is currently under investigation (Turturo et al., 2003). Due to the fact that no function could be assigned to the ORF 2 protein, it could be that this protein plays a role in PTGS suppression, even though it has no sequence homology to any other known proteins. As mentioned previously, in 1999, an infectious clone of GVA isolate PA3 was constructed by Galiakparov et al. An infectious clone is a full-length cDNA clone of a viral RNA genome that can be transcribed, either in vitro or in vivo. Infectious clones can aid as a tool for the investigation and modification of RNA viral genomes at a molecular level. They can facilitate studies of viruses whose isolations are problematic and viruses that are present only in very low titers. They can also provide useful information in the study of the genetic expression and replication of RNA viruses by the use of deletions, insertions, mutagenesis and complementation studies. Furthermore it can aid in the study of natural or induced RNA recombination, plant-virus movement such as the mechanisms of cell-to-cell movement and pathogen-host interactions. These clones can also be considered as viral gene pools for the design of antiviral strategies, trans-complementation studies and the development of new viral vectors. There are a few limitations and pitfalls when it comes to the construction of infectious clones. Furthermore there are factors that strongly influence infectivity of the infectious clone (Boyer & Haenni, 1994). The construction of an infectious clone of GVA in South Africa, will supply us with a tool to investigate the genome of GVA. The GVA genome will be represented in complementary DNA (cDNA) form, which will allow us to study the genome organization and replication at a molecular level. Mutation analysis (introducing mutations into all ORFs) will shed light on gene expression of 5.

(20) GVA. RNA, produced in vitro from the cDNA clone, could be used to infect N. benthamiana plants directly, which could lead to the study of plant-pathogen interactions at a molecular level. This could ultimately lead to the unravelling of the aetiology of Shiraz disease, which could lead to the elucidation of this destructive disease in South Africa.. 2.2. GRAPEVINE VIRUS A 2.2.1. Genus Vitivirus The genus Vitivirus is one of 8 genera in the family Flexiviridae. It contains viruses that were originally tentatively included in the genus Trichovirus. The genus name is derived from the host of the type member, Vitis vinifera (www.dpvweb.net/notes/showgenus.php?genus=Vitivirus). Grapevine virus A is a member of this recently established genus Vitivirus which includes four defined species: GVA, Grapevine virus B (GVB), genus Vitivirus, family Flexiviridae, Grapevine virus D (GVD), genus Vitivirus, family Flexiviridae, and Heracleum latent virus (HLV), genus Vitivirus, family Flexiviridae. A tentative species of this genus is Grapevine virus C (GVC), genus Vitivirus, family Flexiviridae (Martelli et al., 1997). The complete genome sequences are known for both GVA and GVB (Saldarelli et al., 1996; Minafra et al., 1994; 1997) and only partial sequences are available for GVD (AbouGhanem et al., 1997) and HLV. The genomes of vitiviruses have five ORFs, one of which is downstream of the CP cistron (www.dpvweb.net/notes/showgenus.php?genus=Vitivirus). 2.2.2. Morphology Grapevine virus A is a phloem-limited virus, which has flexuous, non-enveloped, filamentous particles of 800 nm in length and 12 nm in diameter (Minafra et al., 1997) (figure 2.1). The nucleocapsid is transversely cross-banded, obliquely striated, and has rope-like features. The virions contain approximately 5 % nucleic acid (www.dpvweb.net/dpv/showdpv.php?dpvno=383).. 6.

(21) Figure 2.1. Electron micrograph of GVA showing its flexuous, filamentous particles. Bar represents 100nm. (www.dpvweb.net /dpv/showfig.php?dpvno=383&figno=06).. 2.2.3. Genome and Genomic organization Grapevine virus A has a monopartite, linear, positive sense single-stranded (ss) RNA genome of 7349 nucleotides (nt), excluding the 3’ poly-A tail. It also possesses a 5’ methylated nucleotide cap. The complete nucleotide sequence of a GVA isolate has been determined (Minafra et al., 1994, 1997). The GVA genome is translated by means of five ORFs into a single protein species of Mr c. 22,000 (www.dpvweb.net/dpv/showdpv.php?dpvno=383) and two (recently identified) nested sets of subgenomic RNAs (Galiakparov et al., 2003) (section 2.2.7.2). The roles of some of the ORFs have been deduced based on sequence homology to known genes (Minafra et al., 1997). In 1999, the construction of an infectious clone of GVA isolate PA3 was reported by Galiakparov et al. (section 2.3.2.1). Mutation analysis was performed on the clone to experimentally define the role of every GVA gene. Putative translation products were assigned for every ORF except for ORF 2 (Galiakparov et al., 2003) (figure 2.2). In 2002, epitope mapping of the GVA capsid protein was performed (Dell’Orco et al., 2002). Results suggested that GVA particles carry a highly structured epitope centered on a common peptide region of the CP sequence.. 7.

(22) Figure 2.2. Genome organization of GVA depicting the five ORFs and their position in the genome. Grapevine virus A has a monopartite, linear, positive sense single-stranded (ss) RNA genome of 7349 nucleotides (nt), excluding the 3’ poly-A tail. It also possesses a 5’ methylated nucleotide cap. The genome is translated by means of five ORFs (mtr – methyl transferase, p-pro – potential product, hel – helicase, pol – RNA-dependent RNA polymerase) (Minafra et al., 1994, 1997).. 2.2.4. Transmission As GVA' s name implies, it is a pathogen of grapevine from which it can be transmitted with difficulty by sap inoculation to a very narrow range of herbaceous hosts. Grapevine virus A is in fact the first phloem-limited virus to be transmitted by sap inoculation to herbaceous hosts. (Conti et al., 1980) (fig. 2.3) It can be transmitted to N. clevelandii and N. benthamiana plants (Galiakparov et al., 1999) where most of the known virus isolates induce systemic vein clearing in 10-12 days, deformation of the leaves and dwarfing (fig. 2.3) (www.dpvweb.net/dpv/showdpv.php?dpvno=383). It is spread by the propagation. of. infected. material. over. medium. and. long. distances. (www.dpvweb.net/dpv/showdpv.php?dpvno=383) and is naturally transmitted between plants by species of the pseudococcid mealybug genera Pseudococcus and Planococcus. (Engelbrecht & Kasdorf, 1997; Garau et al., 1995; Rosciglione et al., 1983). 8.

(23) 2.2.5. Diseases and Geographical distribution Grapevine virus A’s involvement in grapevine diseases is still not clear. It was reported more than 20 years ago (Conti et al., 1980), and it is one of the most frequently detected viruses in vineyards worldwide. Figure 2.3. Stunting and systemic mottling in GVA- infected Nicotiana benthamiana. Healthy plant on the right. (www.dpvweb.net/dpv/showfig.php?dpvno=383&figno=03, DPV383 Figure 03). (Goszczynski & Jooste, 2003). Grapevine virus A probably occurs wherever V. vinifera is grown. It has been detected worldwide from Europe, the Mediterranean basin, Middle East, South Africa, China, Australia, North and Latin America (Boscia et al., 1997a). Results obtained in different laboratories worldwide suggest that GVA is involved in the aetiology of Kober stem grooving (Digiaro et al., 1994; Chevalier et al., 1995; Garau et al., 1995), which is one of the four economically important diseases of the grapevine rugose wood complex (Martelli, 1993). The virus is responsible for crop losses from 5 to 22 % in wine grape cultivars in Italy (Gurau et al., 1997), and for decline and death of table grapevines affected by leafroll disease (Digiaro et al., 1997). As mentioned previously, GVA is important in South African vineyards for its role in grapevine leafroll disease and Shiraz disease (Pietersen, G., Pers. Comm., Department of Plant Pathology, University of Pretoria, South Africa).. 9.

(24) 2.2.5.1. Shiraz disease and Syrah decline Syrah is the French name for the grapevine cultivar, known commonly as Shiraz, in South Africa. Shiraz disease and Syrah decline are the names given to two distinct new emerging diseases. They can be easily confused with one another. 2.2.5.1.1. Shiraz disease (SD) Shiraz disease is an emerging disease of grapevine in the Western Cape (and elsewhere) with very distinctive symptoms, and a crippling effect in vineyards. This deadly disease infects the noble grapevine cultivars Shiraz and Merlot in South Africa. Infected vines deteriorate relatively fast and normally die within five years. Affected vines are rubbery (do not lignify) and keep their leaves longer than healthy vines. The disease causes delayed budding in Merlot. The aetiology of the disease is largely unknown, but is believed to be of multiple viral origin with GVA consistently associated with the disease (Goszczynski, D., Pers. Comm., Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). Burger & Spreeth (1993) reported that 50 SD-affected grapevines were infected with GLRaV-3 and/or GLRaV-2 or GVA using ELISA and immuno-electronmicroscopy (IEM). Goszczynski et al., (2003) re-examined their findings by RT-PCR. Results showed that 91 SDaffected grapevines cv. Shiraz and 35 SD-affected grapevines cv. Merlot were all infected with GLRaV-3 and GVA. Further results suggested that only GVA is required for inducing the disease, although detection of both GLRaV-3 and GVA suggested involvement of both viruses in SD. They also suggested that a certain concentration of GVA may be necessary to induce Shiraz disease and furthermore that a specific group of GVA variants may be associated with Shiraz disease. The successful transmission of SD by the mealybug Planococcus ficus, strengthens the argument that GVA may be the causative agent. GLRaV-3’s involvement in SD could be accidental, as this virus is very common in vineyards in the Western Cape (Goszczynski, D., Pers. Comm., Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). GLRaV-3 may also be required for the efficient transmission of GVA by mealybugs (Engelbrecht & Kasdorf, 1990a; La Notte et al., 1997).. 10.

(25) 2.2.5.1.2. Syrah decline Syrah decline is a mysterious condition that affects Syrah vineyards in California and France about which very little is known. In France, symptoms include the development of swollen and cracked graft unions (“graft union collapse”) followed by leaf reddening in mid to late summer, and eventual death over a four-to-ten year period. Syrah decline in France has been of concern since the early 1990s, because Syrah is the fourth most important red variety in France constituting 56 000 ha of vineyard in 2002. No causative agents have been identified as yet, although a range of fungi, playing a secondary role in accelerating the decline, including Phomopsis, Verticillium and Alternaria have been associated with the graft unions of symptomatic vines (Stamp, 2004). In California, on the other hand, affected Syrah vines develop red leaves, vines may remain green, graft unions may or may not become cracked and swollen and the disease does not kill the plant. It is still not clear whether Syrah decline in France is related to this similar condition in California, provisionally called Syrah disorder (Stamp, 2004). A possible breakthrough in Syrah decline was made when Dr. Adib Rhowani (Stamp, 2004) determined that specific Syrah decline/disorder symptomatic plants from France and California tested negative for all grapevine viruses except for Rupestris stem pitting virus (RSP), genus Foveavirus, family unallocated. RSP has not yet been thought to be a threat to vineyard productivity and it is widely dispersed worldwide in grapevine plant material. An interesting fact is that studies have shown that the RNA of the RSP strains isolated from Syrah decline-affected vines in both France and California differ substantially from the previously characterized RSP virus. The two RSP strains in France and California, associated with Syrah decline, also substantially differ from each other, but it is not clear whether this genetic sequence difference is sufficient to cause a condition such as Syrah decline or whether it is in any way related to it. It could, in fact be quite possible that an altogether previously unknown and undetected virus is responsible for Syrah decline, because of the presence of uncharacterized genetic material in Syrah decline-affected vines (Stamp, 2004). 2.2.6. Molecular diversity Theoretically, all RNA viruses have the potential to establish very large population diversity, because of their error-prone replication, mutation rates in the range of 10-3–10-5 misincorporations per 11.

(26) nucleotide copied (Smith & Simmonds, 1997; Drake & Holland, 1999), and their short generation times. However, there is little reliable information on polymerase fidelity and generation time of RNA plant viruses, which make diversity estimates difficult. Virus diversity can also be overestimated according to the strategy used for its assessment, due to errors introduced during analysis (Smith et al., 1997). Diversity has been observed between virus genomes within the same individual, with the characterization of a population of closely related variants termed viral quasispecies (Martel et al., 1992; Forns et al., 1999). Viral quasispecies are closely related (but non-identical) mutant and recombinant viral genomes subjected to continuous genetic variation, competition, and selection. Quasispecies structure and dynamics of replicating RNA enable virus populations to persist in their hosts and cause disease (Domingo et al., 1998). Several techniques are used to study diversity. A quasispecies distribution within a population can be studied using a sequence-based approach, a technique that is time-consuming. To obtain results rapidly, some techniques using differential gel electrophoresis mobility can be applied to amplified product, such as temperature gradient gel electrophoresis (Lu et al., 1995), single-strand conformation polymorphism analysis (SSCP) (McKechnie et al., 2001), and heteroduplex tracking analysis (Gretch et al., 1996). Whatever the strategy adopted to study quasispecies distribution within a population of a RNA virus, nucleotide sequence information is obtained through PCR amplification of virus-specific cDNA produced by reverse transcription of viral RNA. The enzymes used through these two steps, a reverse transcriptase and a thermostable DNA polymerase, exhibit relatively high error rates which should be taken into account when considering the actual heterogeneity within a viral population (Malet et al., 2003). Recent studies indicated a correlation between mutation frequency and virus host range, suggesting that diverse populations constitute an advantage for RNA plant viruses. An insect feeding is likely to transmit a virus to a variety of plant hosts and host adaptability could mean survival for a plant virus in a natural setting (Schneider et al., 2000; Schneider et al., 2001; Roossinck, 2003). Recently, singlestrand conformation polymorphism (SSCP) analysis of GVA isolates in South Africa recovered in N. benthamiana, revealed extensive molecular heterogeneity of the virus (Goszczynski & Jooste, 2002). Following this study, Goszczynski and Jooste recovered eight isolates of GVA, which induced different symptoms in leaves of N. benthamiana, from various grapevines. Four kinds of symptoms were induced on leaves: (1) mild vein clearing; (2) vein clearing plus interveinal chlorosis; (3) vein clearing, interveinal chlorosis plus strong curling of top leaves; and (4) extensive “patchy” necrosis. They found 12.

(27) that the dsRNA patterns of two isolates, that consistently induced mild vein clearing (referred as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Their analysis based on overall nucleotide (nt) sequence identity in the 3’ terminal part of the GVA genome, comprising part of ORF 3, entire ORF 4, entire ORF 5 and part of 3’ UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0-99.8% nt sequence identity within groups and 78.0% - 89.3% nt sequence identity between groups. Mild isolates of the virus were located in group III and shared only 78.0% - 79.6% nt sequence identity with the other isolates. Their comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF 5, they designed oligonucleotide primers for simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented by mild isolates of the virus (Goszczynski & Jooste, 2003). 2.2.7. Replication mechanism of GVA Grapevine virus A replicates in the cytoplasm, possibly in association with membranous vesicles (www.dpvweb.net/dpv/showdpv.php?dpvno=383). After the construction of a full-length infectious clone of GVA isolate PA3 had been reported (Galiakparov et al., 1999) (section 2.3.2.1), the clone was utilized in an attempt to experimentally define the roles of various GVA genes. Mutations were inserted into every ORF, and the effect on viral replication, gene expression, symptom expression and viral movement were studied in N. benthamiana protoplasts (Galiakparov et al., 2003). Results showed (figure 2.4) that ORF 1 (nt 87-5210) encodes a 194 kDa polypeptide with conserved motifs similar to replication-related proteins (methyl-transferase, RNA-dependant RNA polymerase and RNA helicase) of the “Sindbis-like” supergroup of positive-strand ssRNA viruses (Minafra et al., 1997). A frameshift mutation, introduced at nucleotide position 3632 of this ORF, abolished RNA replication (Galiakparov et al., 2003). This truncated polypeptide lacked the RNA-dependant RNA polymerase domain located between amino acids 1234 and 1673 (Galiakparov et al., 2003). ORF 2 (nt 5179-5712) encodes a protein of 19 kDa with no significant homology with any other proteins (Minafra et al., 1994). None of the aforementioned parameters were affected after inserting a 28 nucleotide deletion, between two Acc I sites, within ORF 2 (Galiakparov et al., 2003). ORF 3 (nt 5654-6490) encodes a polypeptide of 31 kDa, with amino acid similarity to putative movement proteins (MPs) from the 30 K superfamily (Minafra et al., 1994). A 355 nucleotide GUS gene substitution in ORF 3, and two introduced frameshift mutations in ORF 4 (nt 6414-7010), coding for the 22 kDa CP, restricted viral movement. 13.

(28) This implies that ORF 3 codes for the GVA movement protein (Galiakparov et al., 2003). A mutation introduced in ORF 5 (nt 7015-7281), by changing the start codon to ATC, partially restricted viral movement and rendered the virus asymptomatic. ORF 5 codes for a small 10 kDa protein (putative RNA binding protein) with no homology to other known proteins (Galiakparov et al., 2003). However, the corresponding ORF 5 of the closely related vitivirus GVB shares homology with small, 3’-terminal polypeptides of various plant viruses that contain the zinc finger domain of nucleic acid binding proteins (Minafra et al., 1994; 1997).. Figure 2.4. Replication mechanism of GVA. The translation products of all 5 ORFs are shown and the effect that an introduced mutation would have on viral replication given. ORF 1 codes for a 194 kDa putative replicase, ORF 2 codes for an unknown protein, ORF 3 codes for a putative movement protein, ORF 4 is the CP and ORF 5 codes for a RNA binding protein (Galiakparov et al., 2003).. 14.

(29) 2.2.7.1. Synthesis of Subgenomic RNAs by Positive-Strand RNA Viruses Many RNA viruses encode several genes on a single genomic RNA, but only the first 5’-proximal ORF, on a normal eukaryotic mRNA is translated. Downstream genes on viral genomes are thus expressed either via novel translation events or, more commonly, by deployment of subgenomic mRNAs (sgRNAs). Subgenomic RNAs of positive-strand viruses have the same 3’ ends as genomic RNA, but have deletions at the 5’ ends to bring the 5’ end of the RNA in proximity with the start codon of downstream (on genomic RNA) ORFs. The RNA-dependant RNA polymerase (RdRp) is always translated first, directly from genomic RNA of positive-strand RNA viruses, because translation is required for sgRNA synthesis. sgRNAs express products needed during intermediate and late stages of infection, such as structural or movement proteins (Miller et al., 2000). Understanding mechanisms of sgRNA synthesis is important because it will shed light on how these RNA viruses, and GVA in particular, replicate. It may also provide insight into the origin and diversification of viruses by recombination. 2.2.7.2. Subgenomic RNAs in GVA? A study undertaken by Galiakparov et al. (2003) to unravel the regulation of gene expression and the synthesis of viral sgRNAs in GVA led to the characterization of two nested sets of sgRNAs. They explored the production of viral RNAs in a GVA-infected N. benthamiana herbaceous host. This led to the characterization of one nested set of three 5’-terminal sgRNAs of 5.1, 5.5, and 6.0 kb, and another of three 3’-terminal sgRNAs of 2.2, 1.8, and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Results obtained, suggested that expression of ORF 5 occurred via bi- or polycistronic mRNA, because neither 3’- nor 5’-terminal sgRNAs, which would correspond to this ORF, was detected. The 5’-terminal sgRNAs were abundant in dsRNA-enriched extracts. Cloning and sequence analysis of the 5’ end of the 1.8 kb 3’-terminal sgRNA and the 3’-end of 5.5 kb 5’-terminal sgRNA suggested that a mechanism other than specific cleavage was involved in production of these sgRNAs. Sequences upstream of the 5’-terminus of each of ORFs 2-4 apparently controlled the production of the 5’- and 3’-terminal sgRNAs. Detection of both plus and minus strands of the 5’- and 3’-terminal sgRNAs, though in different levels of accumulation, suggested that each of these cis-acting elements is involved in production of four RNAs: a 3’-terminal plus-strand sgRNA which could act as an mRNA, the corresponding 3’-terminal minus-strand RNA, a 5’-terminal plus-strand sgRNA, and the corresponding 5’-terminal minus-strand (Galiakparov et al., 2003). 15.

(30) 2.2.8. Defective RNAs Some single-stranded positive sense RNA viruses are known to support the replication of smaller-thangenomic defective RNAs (dRNAs) in infected host cells which, when they interfere with viral RNA accumulation and symptom expression, are referred to as defective interfering RNAs (DI RNAs) (Lewandowski et al., 1998; Simon et al., 1994; White et al., 1999). These RNAs are a mosaic of genome fragments originated by deletion or recombination events. A study was undertaken by Obreque et al. (2003) to investigate the possibility that during vitivirus replication, defective RNAs may arise, which contain essentially unmodified 5’- and 3’-ends. Deleted chimaeric RNAs, with conserved 5’ and 3’ ends, resembling defective RNAs were detected in GVA-infected N. benthamiana plants by RTPCR amplification. A first class of molecules about 790 bp in size contained the 5’ UTR and the first 459 nucleotides of ORF 1 fused to a 3’ end region (from position 7010 up to the 3’ end primer sequence) consisting of the whole ORF 5 and the 3’ UTR. Obreque et al. sequenced three clones from this class of molecules, which showed a slight variability (3% average) at the nucleotide level in the coding sequences. A second class of molecules, about 420 bp in size, contained the 5’ UTR, the first 307 nucleotides of ORF 1 and the 3’ end primer, without any residue of ORF 5 and 3’ UTR. Two clones from this class of molecules were sequenced, which again showed minor sequence divergence. What is interesting from these results is the fact that ORF 5 is conserved in the larger GVA dRNA, since this gene does not seem to be expressed via a subgenomic RNA. Its presence as dRNA may account for an involvement of the expression product of this ORF, supposed to be involved in suppression of gene silencing (Galiakparov et al., 2003). Further investigations are required to investigate the influence of sgRNAs and dRNAs on virus replication and accumulation and de novo production in plants. 2.2.9. RNA gene silencing A number of living organisms use a general mechanism of post-transcriptional regulation of gene expression, known as RNA silencing. In plants this has evolved into a defence mechanism against viruses based on target sequence-specific degradation (Voinnet, 2001). Plant viruses, in response, have developed the ability to counteract host-induced silencing by means of proteins encoded in their genomes (Li et al., 2002; Voinnet et al., 1999). Diverse virus species use distinct strategies to target the host gene silencing machinery by expressing structurally and functionally different proteins, which effectively suppress PTGS (Voinnet et al., 2000). Recently, clear-cut PTGS suppression was observed 16.

(31) in N. benthamiana transgenic plants, expressing a GFP transgene, following GVA infection. Identification of the GVA protein(s) determining PTGS suppression is currently under investigation (Turturo et al., 2003).. 2.3. INFECTIOUS CLONES Worldwide viruses cause massive damage to crop quality and yield. There are currently no known resistance genes to grapevine viruses and no cures or treatments against plant viruses exist. By analyzing viral genomes at a molecular level, deeper insight can be gained into their genome organization viral gene function, presence of regulatory sequences (and their function) and gene expression. A positive aspect in this regard is the fact that viral genomes are relatively small, which make them amenable to such investigations by recombinant DNA technology. Molecular analysis of viruses whose replication cycle encompass a DNA intermediate step, are much easier to perform than nonretroviral RNA viruses. Reverse genetics, the ability to produce specific mutations followed by examination of phenotype, revolutionized the study of RNA viruses. The potential of investigations has also greatly been enhanced by the possibility of obtaining infectious clones (as in vitro-transcribed RNA copies or as cDNAs) corresponding to the genomes of RNA viruses (Boyer & Haenni, 1994). An infectious clone is a full-length cDNA copy of a viral genome generated by reverse transcription. This full-length clone can be transcribed in vitro by use of a bacteriophage RNA polymerase (T7, T3 or SP6) or linked to a Cauliflower mosaic virus (CaMV), genus Caulimovirus, family Caulimoviridae, 35S promoter to produce in vivo transcripts (Boyer & Haenni, 1994; Baulcombe et al., 1995; Gal-On et al., 1995; Fakhfakh et al., 1996). Infectious clones have several advantages. They can facilitate studies of viruses whose isolation is problematic or viruses that are present in very low titres. By introducing mutations into the cDNA clone, information can be provided in the study of genetic expression and replication of RNA viruses. Furthermore it can aid in the study of natural or induced RNA recombination, plant-virus movement such as the mechanisms of cell-to-cell movement and pathogenhost interactions. By obtaining cDNA clones of most RNA viruses, viral gene pools will be established for the design of antiviral strategies, trans-complementation studies and the development of new viral vectors (Boyer & Haenni, 1994).. 17.

(32) 2.3.1. Construction of infectious clones There are currently two ways of constructing infectious clones: namely (1) The construction of a fulllength cDNA clone of a viral genome, from which an in vitro infectious transcript can be synthesized under the influence of a bacteriophage (T7, T3 or SP6) RNA polymerase promoter, and (2) The expression of infectious viral RNAs by in vivo transcription of cDNA-containing vectors through a CaMV 35S promoter (Boyer & Haenni, 1994; Baulcombe et al., 1995; Gal-On et al., 1995; Fakhfakh et al., 1996). 2.3.1.1. Full-length cDNA clones There are a few limitations and pitfalls when it comes to the construction of a full-length infectious clone of a viral genome from which an in vitro infectious transcript can be produced. It can be a long and tedious process to construct a full-length clone that is infectious. The possibility of producing infectious transcripts from incomplete viral cDNA clones has been reported, even though the presence of the entire viral sequence is generally, thought to be required to obtain infectious clones, but this does not ensure biological activity (Davis et al., 1989; Klump et al., 1990). In recent studies, it was found that cDNA synthesis, cloning strategies and the design of sequences bordering the viral insert, strongly influences the infectivity of the viral insert (Boyer & Haenni, 1994). 2.3.1.1.1. Basic approach in construction of full-length cDNA clones In general, the construction of a full-length infectious cDNA clone consists of purification of virus from infected material and purification of the viral RNA. By using a primer hybridizing specifically to the 3’ end of the viral genome, the viral RNA is transcribed into single-stranded DNA. The viral RNA is then removed and eliminated. DNA synthesis is initiated with a second primer encompassing the sequence corresponding to the nucleotides at the 5’ end of the viral RNA. The single-stranded DNA is thus converted into double-stranded form. Typically, the recognition sequence for a RNA polymerase promoter fused to the viral sequence, are included in the second primer. The synthesis of the full-length first cDNA strand appears to be a serious limiting factor for some viral genomes, because the polymerization step is hampered by strong secondary structures on the viral template RNA (Boyer & Haenni, 1994). It is difficult to sort out an optimum protocol for any virus, because several variations on the general scheme for construction of full-length cDNA clones have been reported (Boyer & 18.

(33) Haenni, 1994). A trend has developed where researchers use thermostable and high fidelity reverse transcriptases and DNA polymerases, both with proofreading capability, to improve the sequence accuracy of the infectious clone. This will ultimately increase the infectivity of the clone and decrease the number of mutations incorporated during reverse transcription and PCR. Interestingly, it has been reported that PCR with Taq polymerase, can be successfully applied to obtain infectious clones (Hayes & Buck, 1990), despite the high error rate of the enzyme (Keohavong & Thilly, 1989). 2.3.1.2. Infectious cDNAs As stated previously, the expression of infectious viral RNAs by in vivo transcription of cDNAcontaining vectors through a CaMV 35S promoter is the second approach that can be followed when constructing infectious clones or transcripts. 2.3.1.2.1. Advantages This approach has several advantages. Firstly, the replication process can overcome detrimental effects resulting from RNA degradation, because infectivity of the clone is less dependent on RNA degradation since it presumably occurs only within cells where the RNAs are synthesized. Secondly, an in vitro transcription step is not required. This is particularly important for RNA viruses for which the production of a good yield of highly infectious full-length transcripts can be problematic. This is also less expensive, because costly reagents such as the cap analogues and RNA polymerases are not required (Boyer & Haenni, 1994). Lastly, the viral replication process and the expression of viral genes are rendered largely independent of each other, which might be very convenient when studying the role and/or localization of proteins expressed by mutant viral RNAs unable to replicate in cells. In vivoviral transcripts produced in this way would then behave like messenger RNAs produced by a host RNA polymerase, still able to express native or mutant proteins without being replicated (Van Bakoven et al., 1993). 2.3.2. cDNA clones for plant RNA viruses Infectious transcripts for members of most plant virus groups have been generated from cDNA clones of plant RNA viruses using both described methods (section 2.3.1) (Boyer & Haenni, 1994). Among potyviruses for example, transcripts have been synthesized in vitro (T7, T3 or SP6) and proved to be 19.

(34) infectious for among others, Potato virus A (PVA), genus Potyvirus, family Potyviridae (Puurand et al., 1996) and Potato virus Y (PVY), genus Potyvirus, family Potyviridae (Jakab et al., 1997). In vivo (CaMV 35S promoter) infectious transcripts have also been reported for PVY (Fakhfakh et al., 1996; Jakab et al., 1997) among others. For vitiviruses, full-length copies of the genomes of GVA (Galiakparov et al., 1999) and GVB under the control of bacteriophage T7 RNA polymerase promoter have been synthesized and both transcribed cDNAs were infectious when mechanically inoculated to N. benthamiana plants (Saldarelli et al., 2000). Capped in vitro-transcribed RNA was infectious in N. benthamiana and N. clevelandii plants (Galiakparov et al., 1999) (section 2.3.2.1). 2.3.2.1 Infectious RNA transcripts from GVA cDNA clone In 1999, Galiakparov et al. constructed an infectious clone of GVA isolate PA3 (provided by A. Minafra and G.P. Martelli). They partially purified GVA by polyethylene glycol precipitation and extracted RNA from virions. They extracted dsRNA by CFII chromatography followed by CC41 chromotography. Reverse transcription was performed on the dsRNA followed by PCR. Fragments were cloned into vectors and the full-length clone was constructed with restriction digestion. A T7 promoter was incorporated immediately adjacent to the 5’-end of the GVA genome and a poly-A tail adjacent to the 3’-end. After in vitro transcription, resulting RNA was used to mechanically inoculate N. benthamiana and N. clevelandii plants. After 10-14 days, the symptoms induced by the RNA transcripts were indistinguishable from the parental virus. Observation of the virions by electron microscopy and serological detection of the virus coat and movement proteins were used to confirm the infectivity of the in vitro-produced transcripts. This was the first report of infectious RNA transcripts derived from a full-length clone of a member of the Vitivirus genus (Galiakparov et al., 1999). 2.3.3. Factors influencing infectivity of the cDNA clone There are a few factors that influence the infectivity of an infectious clone: namely (1) the heterogeneity of transcript population, (2) the presence of point mutations, (3) presence of nonviral nucleotides, (4) Instability in bacteria, and (5) RNA polymerase used.. 20.

(35) 2.3.3.1. The heterogeneity of transcript population Heterogeneity of transcript size was reported to be a problem when using the E. coli RNA polymeraseλPm promoter system (Ahlquist et al., 1984; Dawson et al., 1986; Janda et al., 1987; Hamilton & Baulcombe et al., 1989). Competition between incomplete non-replicatable viral copies and full-length transcripts for interaction with viral and/or host factors involved in the replication process, could account for the relatively low infectivity of most preparations. This problem can be circumvented by using a bacteriophage promoter (Boyer & Haenni, 1994). The relative low fidelity of most reverse transcriptases and polymerases, increase the amount of misincorporations into the genome and contribute to heterogeneity. 2.3.3.2. The presence of point mutations When working with long viral genomes, like GVA, point mutations are to be expected. This is mainly due to the relatively poor fidelity of the RNA- and DNA-synthesizing enzymes. Furthermore, the low fidelity of viral dependent RNA polymerases can lead to the faithful reverse transcription and amplification of an initial virion RNA, which is mutated (lethal mutant) and would most probably be eliminated in the next round of viral replication. The resulting full-length cDNA clone, or the in vitro transcript, would not be infectious. It has been reported that in some cases infectivity can be restored by exchanging a specific region of the cDNA with a fragment corresponding to the same region from an independent cDNA clone (Ahlquist et al., 1984). Conversely, possible mutations favouring infectivity of the full-length transcripts may occur, as it has been reported that certain synthetic transcripts induced more severe symptoms than the parental virion RNAs (Hamilton & Baulcombe, 1989, Hayes & Buck, 1990). As mentioned previously, it has been reported that PCR with Taq polymerase, can be successfully applied to obtain infectious clones (Hayes & Buck, 1990), despite the high error rate of the enzyme (Keohavong & Thilly, 1989). 2.3.3.3. Presence of nonviral nucleotides Many authors have studied the effect of nonviral nucleotides at the extremities of viral transcripts. It is generally admitted that extensions at the 5’-end of viral transcripts, strongly reduces infectivity, whereas 3’-extensions are more easily tolerated (Boyer & Haenni, 1994). 21.

(36) 2.3.3.3.1. Effect of 3’-extensions Different studies performed on the effect of 3’-extensions, revealed the following facts (Boyer & Haenni, 1994). Short 3’-extensions of 1-7 nucleotides don’t seem to influence the biological activity of viral transcripts (Dawson et al., 1986), whereas long extensions between 82 (Dzianott & Bujarski, 1989) and 2700 nt (Ahlquist et al., 1984) for different viruses abolishes infectivity. For many viruses, transcripts bearing a long (>30 nt) additional sequence at the 3’-end are infectious (Dzianott & Bujarski, 1989), as are in vivo transcripts presumably polyadenylated by host cell enzymes. It is not clear whether the structure of additional 3’-sequences affects the biological activity of the transcripts as Sarnow (1989) showed that 4 extra bases (after a short poly-A tail) at the 3’-end of synthetic poliovirus transcripts did not lower infectivity, but 17 cytosine residues decreased infectivity. The same study revealed that the presence of long homopolymeric adenine sequences (poly-A tail) at the 3’-end of in vitro-produced poliovirus transcripts increases infectivity, whereas an adverse effect is seen for long heteropolymeric nucleotide sequences (Sarnow et al., 1989). Dzianott and Bujarski (1989) reported that infectivity of Brome mosaic virus (BMV), genus Bromovirus, family Bromoviridae, transcripts presenting 19 extraviral nucleotides was higher than those harboring 6 or 7 nonviral nucleotides at the 3’-end. This could be due to the fact that longer extensions could confer protection in vivo against ribonucleases, or due to the fact that it could alternatively modify the 3’-end. This modification could cause the 3’-end to acquire an enhanced affinity for the BMV replicase (Dzianott & Bujarski, 1989). 2.3.3.3.2. Effect of 5’-extensions Different studies performed on the effect of 5’-extensions, revealed the following facts (Boyer & Haenni, 1994). In most cases, infectivity is greatly decreased for 5’-extensions, even for only 1 or 2 nucleotide extensions. When transcripts derived from plant viruses harbor moderately long 5’ additional sequences of 14 to 17 nucleotides (Heaton et al., 1989), infectivity is completely abolished. Commandeur et al. (1991) made an interesting observation when cloning cDNA sequences of Beet necrotic yellow vein virus (BNYVV), genus Benyvirus, family unallocated, downstream of the CaMV 35S promoter. The resulting in vivo transcripts, containing up to 40 extra-viral nucleotides at the 5’end, were infectious in planta. In vitro-derived transcripts containing the same extensions, were biologically inactive in the host plant. Another interesting fact is that the infectivity of animal viruses, compared to plant viruses, is less affected when transcripts contain relatively large 5’-extensions. It is assumed that the problem with extra 5’-non-viral sequences is that its presence could seriously hamper 22.

(37) proper initiation of (+) RNA synthesis from the 3’-end of the (-) strand. These nucleotides do not possess initiation codons, so it seems unlikely that they interfere with in vivo viral gene translation, and the in vitro translation products are similar to those of wild-type RNA (Janda et al., 1987; Verver et al., 1987; Angenent et al., 1989; Eggen et al., 1989; Dore et al., 1990; Gal-On et al., 1991; Viry et al., 1993). 2.3.3.3.3. Effect of the cap structure Studies performed on the effect of the cap structure (for viruses without a viral genome linked protein, VPg), revealed the following facts (Boyer & Haenni, 1994). For optimum infectivity, as a general rule, a cap structure (m7GpppG) is required at the 5’-end, possibly because it enhances translation initiation (Shih et al., 1976) and/or improves their stability by conferring a better resistance to host cell nucleases (Furuichi et al., 1977). In a few cases both capped and uncapped transcripts proved to be infectious, although uncapped transcripts were almost always either not infectious or had a highly reduced level of infectivity (Angenent et al., 1989). 2.3.3.4. Instability in bacteria Another limiting factor is the high instability of full-length cDNA clones in bacteria. This problem is especially manifested in (+) RNA viruses (of which GVA is one). As a consequence, infectious clones for some viruses are either impossible to assemble, or once assembled, are very difficult to maintain in E. coli due to their predisposition to spontaneous rearrangements and/or to acquisition of stabilizing mutations (Yamshchikov et al., 2001). One plausible explanation is that the instability results from unanticipated expression of viral cDNA resulting in products that are toxic to the bacterial host. While stable infectious clones for some viruses have been reported (Boyer & Haenni, 1994; Khromykh & Westaway, 1994; Meyers et al., 1996; Geigenmuller et al., 1997), similar constructs for other viruses exhibit acceptable stability only after assembly in low copy number vectors (Gritsun & Gould, 1998; Gualano et al., 1998; Mendez et al., 1998; Hurrelbrink et al., 1999; Almazan et al., 2000). In order to circumvent the instability problem for certain viruses, more sophisticated procedures have been used for assembly of infectious clone cDNA templates. Methods used include, in vitro ligation (Sumiyoshi et al., 1992; Kapoor et al., 1995), long high-fidelity PCR (Campbell & Pletnev, 2000), or a combination of both (Herold et al., 1998), the insertion of short introns into problematic expression regions (Yamshchikov et al., 2001), and the introduction of frameshift mutations in infectious cDNA 23.

(38) clones (Satyanarayana et al., 2003). The use of nonbacterial cloning systems (Polo et al., 1997) has been reported as well. 2.3.3.5. RNA polymerases The choice of the RNA polymerase promoter is important when dealing with the design of a full-length clone from which infectious RNAs are expected to be produced, in vitro or in vivo, because it directly affects the yield of transcripts and the nucleotide sequence at the extremities. Allthough several types of promoters have been used such as the E. coli Pm promoter, from bacteriophage λ (Ahlquist & Janda 1984), and the promoters of bacteriophages SP6 (Melton et al., 1984), T3 and T7 (Dunn & Studier, 1983). The bacteriophage T7 promoter is more commonly used, because of the more thoroughly studied genetics of bacteriophage T7. Compared to phage RNA polymerase-based systems, the the E. coli RNA polymerase-based system produces much lower transcript yields, because it leads to a large proportion of premature termination products (Melton et al., 1984; Janda et al., 1987; Angenent et al., 1989; Heaton et al., 1989). 2.3.4. Conclusion Even though GVA was reported more than 24 years ago (Conti et al., 1980), the surface of GVA’s involvement in disease hasn' t even been scratched. Grapevine virus A is one of the most frequently detected viruses worldwide (Goszczynski & Jooste, 2003) and cause dramatic losses to vineyards worldwide. In South Africa (and elsewhere), a new disease known as Shiraz disease is threatening to become a problem. Since GVA is thought to be associated with the disease (Goszczynski & Jooste, 2003), the construction of an infectious clone of GVA could greatly benefit in unravelling the aetiology of the disease. Infectious cDNA clones of RNA viral genomes have been constructed for many viruses. They serve as tools to study RNA viral genomes at a molecular level. There are many pitfalls when it comes to the construction of such clones. When these pitfalls are overcome, infectious clones can lead to the unraveling of genome organization, gene expression and pathogen-host interactions of RNA viruses. This could ultimately lead to development of resistance to, and proper control over, disease.. 24.

(39) Chapter 3: Materials and Methods 3.1. PLANT MATERIAL 3.1.1. Plant material Genomic fragments of GVA were obtained from two different plant sources, namely GVA infected grapevine, I 3973 (obtained from Mr. Nolan Africander, South African Agricultural, Food, Quarantine & Inspection Services, Stellenbosch, South Africa), and GVA (GTR1-2) infected N. benthamiana (obtained from Dr Dariusz Goszczynski, Agricultural Research Council Plant Protection Reseach Institute, Pretoria, South Africa). 3.1.2. Plant cultivation N. benthamiana seeds were germinated on damp filter paper in Petri dishes in the dark. Plants were grown in heat sterilized soil in a growth room with controlled conditions of, temperature between 18ºC and 24ºC, relative humidity of approximately 70% and a 16 hour to 8 hour light/dark cycle (Freeborough, 2003). Plants were fed with SEAGRO. (Premier Fishing SA (Pty) Ltd) every two. weeks according to the manufacturer' s instructions.. 3.2. GENERATION OF GVA cDNA FRAGMENTS GVA cDNA fragments were generated with three different methods: namely (1) double stranded RNA extraction followed by 2-step RT-PCR, (2) total RNA extraction followed by 2-step RT-PCR, and (3) rapid direct-one-tube RT-PCR. 3.2.1. Double stranded RNA (dsRNA) extraction: CFII cellulose method Double stranded RNA extraction was performed according to the method of Rezaian and Krake (1987). Grapevine petioles and bark scrapings were prepared for dsRNA extraction and stored at -80°C. Ten grams of phloem tissue was ground to a fine powder in liquid nitrogen, using a sterile mortar and pestle. Ground material was resuspended in 112 ml extraction buffer (45 ml 2x STE [1x STE (100 mM NaCl, 50 mM Tris-HCl pH 7, 1 mM EDTA)], 1.3% SDS, 32 mg Bentonite, 15 mM β-ME, 25 ml 25.

No results found